Phosphatidyl Inositol kinases ("PtdIns-kinases") regulate diverse cellular processes including cell signaling, cell cycle progression, and intracellular protein sorting. See, e.g., Herman et al., Nature 358:157-159 (1992), Kapellar et al., Bioessays 16:565-576 (1994), Stephens et al., Nature 351:33-39 (1991) and Kunz et al., Cell 73:585-596 (1993). PtdIns-kinases phosphorylate phosphoinositol lipids at distinct positions on the inositol ring. For example, PtdIns 3-kinases, or "PI 3-kinases", phosphorylate the D3 hydroxyl group on the inositol ring, while PtdIns 4-kinases phosphorylate the D4 hydroxyl group. All of the PtdIns kinases that have been identified to date have been found to contain a core region of sequence similarity, which, without being bound to any particular theory, is believed to be the catalytic domain. This domain, termed the "PtdIns kinase domain", shares limited sequence similarity with the catalytic domain of protein kinases and mutation of conserved residues results in loss of PtdIns kinase activity. See, Carter et al., J. Biochem. 301:415-420 (1994), Dhand et al., EMBO J. 13:522-533 (1994) and Schu et al., Science 260:88-91 (1993).
A number of receptor tyrosine kinases, src-like tyrosine kinases and viral oncoproteins bind and activate one particular cellular PtdIns 3-kinase. Studies of mutants that abrogate the binding of this PtdIns 3-kinase to these molecules indicate that PtdIns 3-kinases mediate mitogenic and cell motility responses of cells to growth factors and oncoproteins. Purification of the polypeptide subunits of this PtdIns 3-kinase reveal that the enzyme exists as a heterodimeric complex composed of a 110 Kd and an 85 Kd subunit. Carpenter et al., J. Biol. Chem. 265:19704-19711 (1990), Fry et al., Biochem. J. 288:383-393 (1992), Morgan et al., Eur. J. Biochem. 191:761-767 (1990), Shibasaki et al., J. Biol. Chem. 266:8108-8114 (1991). The 110 Kd subunit contains a C-terminal PtdIns kinase domain, as well as a small domain at its N-terminus that is sufficient for binding to the 85-Kd subunit (Hiles et al., Cell 70:419-429 (1992), Holt et al., Mol. Cell. Biol. 14:42-49 (1994), Klippel et al., Mol. Cell. Biol. 14:2675-2685 (1994). The 85 Kd subunit serves as an adapter and binds activated growth factor receptors and other tyrosine phosphorylated molecules through two Src homology 2 (SH2) domains. Hu et al., Mol. Cell. Biol. 12:981-990 (1992), McGlade et al., Mol. Cell. Biol. 12:991-997 (1992) Reedijk et al., EMBO J. 11:1365-1372 (1992), Yoakim, J. Virol. 66:5485-5491 (1992), Yonezawa et al., J. Biol. Chem. 267:25958-25966 (1992). The association of the enzyme with activated growth factor receptors is believed to localize it to the plasma membrane where its phospholipid substrates reside.
The p110/p85 complex phosphorylates distinct lipids in vitro and in vivo. In vitro, the p110/p85 complex can phosphorylate phosphatidylinositol (PtdIns), phosphatidylinositol 4-phosphate (PtdIns4P) and phophatidylinositol 4,5-bisphosphate (PtdIns(4,5)P.sub.2) on the D3 hydroxyl group of the inositol ring, producing phosphatidylinositol 3-phosphate (PtdIns3P), phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P.sub.2) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P.sub.3). Kapellar et al., Bioessays 16:565-576 (1994), Stephens et al., Biochim. Biophys. Acta 1179:27-75 (1993). Activation of the p110/p85 complex in cells results in elevated levels of PtdIns(3,4)P.sub.2 and PtdIns(3,4,5)P.sub.3, but not PtdIns3P. Auger et al., Cell 57:167-175 (1989), Stephens et al., Nature 351:33-39 (1991), Traynor-Kaplan et al., Nature 334:353-356 (1988), Traynor-Kaplan J. Biol. Chem. 264:15668-15673 (1989). Studies of the pathways in cells that generate these lipids suggest that PtdIns(3,4)P.sub.2 is formed by the dephosphorylation of PtdIns(3,4,5)P.sub.3 by a PtdIns 5-phosphatase rather than the phosphorylation of PtdIns4P by a PtdIns 3-kinase. Carter et al., Biochem. J. 301:415-420 (1994), Hawkins et al., Nature 358:157-159 (1992).
The stimulation of cells with growth factors or tumor antigens results in a rapid increase in the cellular concentration of PtdIns(3,4)P.sub.2 and PtdIns(3,4,5)P.sub.3 from a low basal level, indicating that these molecules represent novel second messengers in growth factor activated signaling cascades.
Additionally, several protein kinases have recently been described which appear to represent downstream effectors of the lipid products of PtdIns 3-kinases. The Akt protein kinase can be activated in vivo by treating cells with the mitogen PDGF. The binding of PtdIns 3-kinase to the PDGF receptor is essential for Akt activation (Franke et al., Cell 81:727-736 (1995)). Akt can be directly activated in vitro with PtdIns3P. In addition to Akt, particular isoforms of protein kinase C can be activated by the lipid products of PtdIns 3-kinases. Protein kinase C isoforms .delta., .epsilon., .eta., and .zeta. can be activated in vitro with PtdIns(3,4)P.sub.2 or PtdIns(3,4,5)P.sub.3, but not with PtdIns3P. Nakanishi et al., J. Biol. Chem. 268:13-16 (1993), Toker et al., J. Biol. Chem 269:32358-32367 (1994).
PtdIns 3-kinases have also been implicated in the regulation of intracellular protein sorting. For example, the Vps34 PtdIns 3-kinase was initially identified from a Saccharomyces cerevisiae mutant that was defective in the trafficking of proteins to the lysosome-like vacuole. Herman et al., Mol. Cell. Biol. 10:6742-6754 (1990). Vps34 can phosphorylate PtdIns, but not PtdIns4P or PtdIns(4,5)P.sub.2 in vitro, which is consistent with absence of detectable PtdIns(3,4,5)P.sub.3 in yeast. Schu et al., Science 260:89-91 (1993). Vps34 is the major PtdIns 3-kinase in yeast. VPS34 mutant strains contain no detectable PtdIns3P.
Because disregulation of the cellular processes, such as signaling processes involved in cell cycle progression and intracellular protein sorting, can have disastrous effects, it is important to understand and gain control over these processes. This requires identifying the participants in the signaling events involved in these processes and elucidating their mechanism of function. The identification of these participants is important for a wide range of diagnostic, therapeutic and screening applications. In particular, by knowing the structure and function of a particular participant in a signaling cascade, one can design compounds which affect that cascade, to either activate an otherwise inactive pathway, or inactivate an overly active pathway. Similarly, having identified a particular participant in a signaling cascade, one can also identify situations where that cascade is defective, resulting in a particular pathological state.
PI3-kinases have been identified as playing critical roles in several distinct cellular signaling processes. As a result, it is of particular interest to identify these kinases, their substrates, products and effectors. Once identified, these various elements can be used for a variety of therapeutic, diagnostic and screening applications. For example, these components may be used as therapeutic agents for treating disorders resulting from anomalies in signaling cascades. Alternatively, these components may be used alone or in combination, as model systems for screening compounds that affect signaling cascades. Finally, identification of these components leads to an understanding of the cell signaling processes, anomalies in these processes which are responsible for certain disorders, and by implication, diagnostic systems for identifying these disorders. The present invention meets these and many other needs.